Initial assessment of the spatial learning, reversal, and sequencing task capabilities of knock- in rats with humanizing mutations in the Aβ-coding region of App

Model organisms mimicking the pathogenesis of human diseases are useful for identifying pathogenic mechanisms and testing therapeutic efficacy of compounds targeting them. Models of Alzheimer’s disease and related dementias aim to reproduce the brain pathology associated with these neurodegenerative disorders. Transgenic models, which involve random insertion of disease-causing genes under the control of artificial promoters, are efficient means of doing so. There are confounding factors associated with transgenic approaches, however, including target gene overexpression, dysregulation of endogenous gene expression at transgenes’ integration sites, and limitations in mimicking loss-of-function mechanisms. Furthermore, the choice of species is important, and there are anatomical, physiological, and cognitive reasons for favoring the rat over the mouse, which has been the standard for models of neurodegeneration and dementia. We report an initial assessment of the spatial learning, reversal, and sequencing task capabilities of knock-in Long-Evans rats with humanizing mutations in the Aβ-coding region of App, which encodes amyloid precursor protein (Apph/h rats), using the IntelliCage, an automated operant social home cage system, at 6-8 weeks of age, then again at 4-5 months of age. These rats were previously generated as control organisms for studies on neurodegeneration involving other knock-in rat models from our lab. Apph/h rats of either sex can acquire place learning and reversal tasks. They can also acquire a diagonal sequencing task by 6-8 weeks of age, but not a more advanced serial reversal task involving alternating diagonals, even by 4-5 months of age.


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Technical innovation has enabled researchers in the past two decades to study 37 neurodegenerative disorders with greater precision. For example, optogenetics, a technique for 38 modulating individual neuron activity through activation of light-sensitive proteins called opsins(1), has 39 been used to evaluate grafts of mesencephalic dopaminergic neurons derived from human embryonic 40 stem cells in a mouse model of Parkinson's disease(2); while electron cryo-microscopy, whose 41 resolution has become comparable to that of X-ray crystallography(3) has revealed the structure of and second pass through the program timeline, respectively), as outlined in Table 1

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Nosepoke adaptation (both cohorts, 1 day) -The rats learn they must activate a nosepoke sensor to 151 open a water access door at any corner for seven seconds; this nosepoke mechanic remains active 152 for every program hereafter.

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Time adaptation (Cohort A: 4 days, Cohort B: 2 days) -The rats may only drink between 8pm and 154 11pm at any corner, a time window called the drinking session.
Single corner restriction (Cohort B only, 2 days) -All rats must drink from a single correct corner with 156 the other corners being neutral during the drinking session. The correct corner changes after ninety 157 minutes, such that the rats can drink at one corner during the first half of the drinking session and 158 must switch to the opposite corner during the second half. Over two days, the correct corner 159 designation follows the path 1->3 (1st day), then 2->4 (2nd day), covering all corners ( Figure 2A).

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Place learning (Cohort A only, 3 days) -The rats may only drink during the drinking session at a 161 corner assigned to each of them; these assigned corners are considered correct, and the non-162 assigned corners are considered incorrect ( Figure 2B).

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Place learning with corner switch (Cohort B only, 4 days) -Each rat is assigned an initial correct 164 corner where it can drink during the drinking session, as in place learning, with the other corners 165 being incorrect. Every 45 minutes, the correct corner designations are switched according to the cycle

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If corner 2 were the initial correct corner, the cycle would be shifted over once (2->1->3->4[->2]). After 168 the first switch, the positions of the incorrect corners adjust accordingly. By the first 45-minute block of 169 the next drinking session, the correct corner will have returned to its initial location. A phase refers to 170 a 45-minute block during the drinking session in this program. The end of a phase marks when a 171 corner switch occurs ( Figure 2C).

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Place reversal (Cohort A only, 3 days) -The rats may only drink during the drinking session at the 173 corner diagonally opposite the one assigned in place learning; those reversed corners are considered 174 correct, and the remaining corners, including the original assigned corner, are considered incorrect 175 ( Figure 2D).

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Behavioral sequencing (Cohort A: 3 days, Cohort B: 5 days) -The rats must alternate between 177 drinking at the initial learned corner and the opposite corner during the drinking session, so that one 178 corner in the assigned diagonal is active (correct) at a time while the other is inactive (opposite); the 179 conditions of the corners in the assigned diagonals alternate between correct and opposite, with a 180 correct nosepoke triggering a condition switch. Visits to corners in the non-assigned diagonal are 181 considered lateral visits ( Figure 2E).

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App h/h rats do not visit corners more often than other App h/h rats during single corner 231 restriction in cohort B at either 6-8 weeks or 4-5 months of age.

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After IntelliCage adaptation as outlined in Table 1, rats in cohort B were started on the single corner 233 restriction program, which tested whether the animals were able to share this corner equally among 234 themselves for water. The animals were assigned a rank during each 90-minute block of the two 235 drinking sessions (four ranks total) based on visits to the actively correct corner, as described in the 236 methods. The mean rank was used to compare animal activity ( Figure 3). There were no significant 237 differences among male rats during either pass. During the first pass one female rat (Animal 22) had a 238 mean rank significantly lower than that of two other female rats (Animals 16 and 17), but this 239 difference was not observed during the second pass.

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App h/h rats in cohort A can acquire a place learning task by 6-8 weeks of age with session-wise 241 improvement. and red circles, respectively. All data represented as mean ± SEM (*p < 0.05). See Table 2 Figure 3 for single corner restriction, cohort B. A p-value less than 0.05 is considered significant (*p < 0.05).
After IntelliCage adaptation as outlined in Table 1, rats in cohort A were started on the place learning 243 program. Animal activity in this program and subsequent programs was summarized via (1)  incorrect visits flattens, session-wise, signifies task acquisition ( Figure 4A). Analysis of area under the 249 curves (AUC) revealed significant session-wise increases for correct visits (C-AUC) with 250 accompanying decreases for incorrect visits (IC-AUC) for both sexes during the first pass ( Figure 4B).

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During the second pass, there were no significant session-wise differences in C-AUC for either sex,

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and IC-AUC was significantly lower for the 3 rd drinking session compared to the 1 st and 2 nd for females 253 alone ( Figure 4B). For the 2 nd drinking session of the first pass, IC-AUC was significantly lower for 254 males ( Figure 4B). For females, C-AUC was significantly higher for each drinking session of the 255 second pass compared to the first, whereas IC-AUC was significantly lower for the 1 st drinking session 256 during the second pass; there were no significant differences between passes for males ( Figure 4C). passes are indicated by white and red circles, respectively. All data represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, similarly for # p < 0.05, etc.). See Tables 3-5 for statistical analysis. FP = first pass (6-8 weeks), SP = second pass (4-5 months), C = correct (visits), IC = incorrect (visits).
App h/h rats in cohort A can acquire a place reversal task by 6-8 weeks of age with session-wise 259 improvement. After place learning, rats in cohort A were started on the place reversal program,

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which switches the correct corner in place learning to the one diagonally opposing it. Activity curves 261 showed qualitative improvement, like those shown for place learning ( Figures 4A and 5A). There were 262 also significant session-wise increases in C-AUC and decreases in IC-AUC for both sexes during the 263 first pass, with no significant differences seen during the second pass for either sex ( Figure 5B).

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There were significant sex differences seen during the first pass, with C-AUC higher and IC-AUC 265 lower for males for every drinking session, but not during the second pass ( Figure 5B). For females,

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C-AUC was significantly higher during the second pass compared to the first for the 1 st and 3 rd 267 drinking sessions, with the value for the 2 nd drinking session being higher but not reaching 268 significance, while IC-AUC was not significantly different for any drinking session; there were no  Table 5 -Statistical analysis of data shown in Figure 4C for place learning, cohort A, pass comparison. A p-value less than 0.05 is considered significant (**p < 0.01, ***p < 0.001). ns = not significant. significant differences between passes for males ( Figure 5C). day, and visit category. Data points from the first and second passes are indicated by white and red circles, respectively. All data represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, similarly for # p < 0.05, etc.). See Tables 6-8 for statistical analysis. FP = first pass (6-8 weeks), SP = second pass (4-5 months), C = correct (visits), IC = incorrect (visits).

Figure 5B
Two-way RM ANOVA, place reversal, cohort A, second pass  Figure 5B for place reversal, cohort A, second pass (4-5 months). A p-value less than 0.05 is considered significant. ns = not significant.

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of age with session-wise improvement. Rather than progressing from place learning to place 274 reversal as cohort A rats did, cohort B rats were started on the place learning with corner switch 275 program after single corner restriction. This program was designed to be a faster-paced combination 276 of place learning and place reversal, with correct corners switching every 45 minutes within a drinking 277 session. Activity curves show marked differences between passes for both sexes, with curves for 278 correct visits surpassing those for incorrect visits earlier during the second pass; notably, for females, 279 the correct curve surpassed the incorrect curve by the end of the 2 nd drinking session during the 280 second pass, whereas the correct curve never surpassed the incorrect one during the first pass 281 ( Figure 6A). No significant session-wise differences in AUC were seen during the first pass for either 282 sex, but there were significant increases in C-AUC and decreases in IC-AUC during the second pass 283 for both sexes, more pronounced for C-AUC ( Figure 6B). Sex differences were significant for each 284 drinking session during the second pass, with C-AUC higher for females and IC-AUC lower for males 285 ( Figure 6B). For females during the second pass compared to the first pass, C-AUC was significantly 286 higher for females for all but the 1 st drinking session, while IC-AUC was only significantly different for 287 the 4 th drinking session ( Figure 6C). For males during the second pass compared to the first pass, C-

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AUC was significantly higher for all drinking sessions, while IC-AUC was significantly lower for all   Figure 5C for place reversal, cohort A, pass comparison. A pvalue less than 0.05 is considered significant (**p < 0.01, ***p < 0.001). ns = not significant. drinking sessions except the 2 nd ( Figure 6C). Pass comparison of AUCs for activity curves of individual animals by sex, program day, and visit category. Data points from the first and second passes are indicated by white and red circles, respectively. All data represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, similarly for # p < 0.05, etc.).

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After place learning and place reversal (cohort A) or place learning with corner switch (cohort B), we 295 further tested the rats' spatial learning capabilities with a behavioral sequencing program requiring the 296 animals to shuttle between diagonally opposing corners for water access. Visits were categorized as programs. Cohort A rats of both sexes during the first pass showed significant increases in C-AUC 300 and decreases in O-AUC, but no significant changes in L-AUC ( Figure 7B). These changes were 301 consistent during the second pass for females, whereas males no longer showed significant session-

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wise changes in C-AUC ( Figure 7B). There were significant sex differences observed for the 3 rd 303 drinking session of the first pass for C-AUC (higher in males) and L-AUC (higher in females), with no 304 significant differences observed during the second pass ( Figure 7B). For females during the second 305 pass compared to the first, C-AUC was significantly greater for the 1 st drinking session; for males, C-

Figure 7C
Paired t tests, behavioral sequencing, cohort A, pass comparison  Figure 7C for behavioral sequencing, cohort A, pass comparison. A p-value less than 0.05 is considered significant (*p < 0.05, **p < 0.01). ns = not significant.

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App h/h rats in cohort A and B may not be able to acquire a serial reversal task by 4-5 months of 334 age.  Figure 8C for behavioral sequencing, cohort B, pass comparison. A p-value less than 0.05 is considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ns = not significant. complexity to behavioral sequencing by requiring the rats to alternate diagonals after every eight 337 correct nosepokes. For cohort A, qualitatively, activity curves for both sexes did not show much 338 difference between passes or session-wise improvement ( Figure 9A). Session-wise differences in 339 AUC were minimal for both sexes during both passes, with some significant sex differences during the 340 second pass ( Figure 9B). Significant differences between passes were sporadic for females in L-AUC

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(2 nd drinking session) and O-AUC (1 st drinking session), and non-existent for males ( Figure 9C). For 342 cohort B, the activity curves show a possible difference between the first and second pass for males, 343 but no session-wise differences ( Figure 10A). AUC analysis revealed that for males compared to 344 females during the second pass, C-AUC was significantly higher for all drinking sessions, with O-AUC 345 significantly lower for the 3 rd and 4 th drinking sessions ( Figure 10B). For males during the second pass 346 compared to the first, C-AUC was significantly higher for every drinking session, with L-AUC 347 significantly higher for the 4 th drinking session; significant differences between passes were non-348 existent for females ( Figure 10C).   Table 18 -Statistical analysis of data shown in Figure 9B for serial reversal, cohort A, first pass (6-8 weeks). A p-value less than 0.05 is considered significant.

Figure 10B
Mixed-effects analysis, serial reversal, cohort B, first pass  Figure 10B for serial reversal, cohort B, first pass (6-8 weeks). A p-value less than 0.05 is considered significant.

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tended to perform better than females at 4-5 months of age in place learning with corner switch and 361 behavioral sequencing. The results of the single corner restriction program for cohort B suggest that 362 although individual variance exists among the rats, it is small enough that animals can be  Figure 10C for serial reversal, cohort B, pass comparison. A p-value less than 0.05 is considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). ns = not significant. approximated as identical subjects for these IntelliCage experiments. Generating activity curves with 364 aggregate cohort data is one way to reduce the impact of this variance on interpretation of cohort 365 performance. Using AUC as a metric for comparing activity between groups is a natural extension of 366 using linear fits on activity curves to estimate learning rate and takes full advantage of the data 367 volume the IntelliCage offers. Task acquisition can be characterized by performance parameters-in 368 this case, AUC-that are greater or less than the value that would be expected through chance alone, learning programs except serial reversal, suggesting that the program is too complex for the rats to 376 learn by 4-5 months of age. In general, a task that challenges the animals without being impossible to 377 acquire would be ideal for identifying possible cognitive deficits in models of neurodegeneration and 378 dementia. Task acquisition of behavioral sequencing but not serial reversal suggests that a program 379 of intermediate difficulty using a sequence involving all four corners (in clockwise motion, for example) 380 rather than just two in a single diagonal, might be worth testing in future studies. By these measures,

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this study establishes a baseline spatial learning profile for App h/h control rats while providing initial 382 validation of analytic methods exploring aggregate cohort activity and using AUC as a metric for task 383 performance in the IntelliCage.